DataSheet1_Red algae industrial residues as a sustainable carbon platform for the co-production of poly-3-hydroxybutyrate and gluconic acid by Halomonas boliviensis.docx

Polyhydroxyalkanoate (PHA) production using halophilic bacteria has been revisited because less severe operational conditions with respect to sterility can be applied, also alleviating production costs. Halomonas boliviensis was selected because it is a moderate halophile able to grow and attain high poly-3-hydroxybutyrate (P3HB) contents under 5–45 g/L NaCl concentrations, conditions that discourage microbial contamination. Industrial residues of the red alga Gelidium corneum after agar extraction were used as sugar platform to reduce costs associated with the carbon source. These residues still comprise a high carbohydrate content (30–40% w/w) of mainly cellulose, and their hydrolysates can be used as substrates for the bioproduction of value-added products. Preliminary assays using glucose were carried out to determine the best conditions for growth and P3HB production by H. boliviensis in bioreactor fed-batch cultivations. Two strategies were addressed, namely nitrogen or phosphorus limitation, to promote polymer accumulation. Similar P3HB cell contents of 50% (gpolymer/gCDW) and yields YP3HB/glucose of 0.11–0.15 g polymer/g glucose were attained under both conditions. However, higher specific productivities were reached under P-limitation, and thus, this strategy was adopted in the subsequent study. Two organic acids, resulting from glucose metabolism, were identified to be gluconic and 2-oxoglutaric acid. Reducing the oxygen concentration in the cultivation medium to 5% sat was found to minimize organic acid production and enhance the yield of polymer on sugar to 0.20 gP3HB/gglucose. Finally, fed-batch cultivations using G. corneum hydrolysates as the only C-source achieved an overall volumetric productivity of 0.47 g/(L.h), 40% polymer accumulation, and negligible gluconic acid production.

聚羟基脂肪酸酯（Polyhydroxyalkanoate，PHA）的嗜盐菌合成工艺，因可采用对无菌要求更低的操作条件并降低生产成本，再度受到研究关注。本研究筛选得到玻利维亚盐单胞菌（Halomonas boliviensis）作为生产菌株，该菌为中度嗜盐菌，可在5~45 g/L氯化钠浓度下生长并积累较高含量的聚3-羟基丁酸酯（poly-3-hydroxybutyrate，P3HB），此浓度范围可有效抑制杂菌污染。为降低碳源相关成本，本研究以琼脂提取后的红藻石花菜（Gelidium corneum）工业残渣作为糖基底物；该残渣的碳水化合物含量达30%~40%（质量分数），主要成分为纤维素，其水解液可作为增值产物的生物合成底物。为确定玻利维亚盐单胞菌在生物反应器补料分批培养中的最佳生长与P3HB合成条件，本研究首先开展了以葡萄糖为底物的预实验。本研究采用氮限制与磷限制两种策略以促进聚合物积累，两种策略下均实现了50%（聚合物质量/细胞干重（Cell Dry Weight，CDW）质量）的P3HB细胞含量，以及0.11~0.15 g聚合物/g葡萄糖的P3HB/葡萄糖得率（YP3HB/glucose）。但磷限制策略下获得了更高的比生产速率，因此后续研究采用该策略。经鉴定，葡萄糖代谢过程中产生的两种有机酸为葡萄糖酸与2-氧代戊二酸。将培养基中的氧浓度降至5%饱和度时，可显著减少有机酸生成，并将聚合物对糖的得率提升至0.20 g P3HB/g葡萄糖。最后，以石花菜水解液作为唯一碳源的补料分批培养实验，最终获得了0.47 g/(L·h)的总体积生产速率、40%的聚合物积累量，且几乎未检测到葡萄糖酸生成。